Project Summary Defined as the collection of all viruses in circulation, human blood virome is a major component in our body's ecosystem. Using next-generation sequencing (NGS) technology, an entire virome can be deciphered without the need of prior sequence information. Such an approach is thus extremely valuable in translational studies, such as transfusion safety, disease etiology, and clinical diagnosis of blood-borne viruses like hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). However, the progress in these fields has been halted by a technical barrier, the low sensitivity in the detection of a human virome owing to excessive human genome contents in blood. As a consequence, virome occupies only a tiny portion of the NGS data output, commonly less than 5%. In HCV patients with viral titers below 105 copies/mL, the virus is hardly detectable by meta-transcriptome sequencing. A low sensitivity on virome detection also aggravates the contamination in NGS in which reagent or environmental contaminants have been mistakenly assigned as novel viruses. Solutions to enhance the sensitivity of virome detection have been explored from both human (host) and viral sides, such as the depletion of selective human genes, capture sequencing with individual or pan-virus probes, and device-based virus enrichment. However, these methods have not been widely adopted perhaps due to laborious, costly and suboptimal procedures. Here, we have proposed a new approach for enhanced blood virome sequencing, which combines our recent invention of a template-dependent multiple displacement amplification (tdMDA) (BioTechnique 2017; Patent: WO 2016164259 A1), emulsion droplets, and a novel depletion strategy. After tdMDA amplification in microfluidic droplets, we hypothesize that a virome could be enriched by the depletion of human genome sequences via duplex-specific nuclease (DSN)-mediated digestion with an excessive 120-bp human shotgun library. As viral genomes are far larger than 120 bp, they remain in the product after size-exclusion by AMPure beads. Using mock samples containing the spike-in HCV genome in the background of human samples, we will first develop and optimize an experimental protocol for enhanced virome sequencing, which will then be verified in thirty well-characterized serum samples from a completed clinical trial. The entire pipeline for enhanced virome sequencing is simple, scalable, and affordable for most clinical and molecular laboratories, and thus represents a desirable tool in the field of human virome research.